We will use in vitro reconstitution assays to identify the function of each protein involved in vesicular stomatitis virus RNA synthesis. Different phosphorylated forms of the NS transcription protein will be analyzed by peptide mapping and tested for their ability to support RNA synthesis in vitro. The polymerase binding site will be defined by nuclease sensitivity and footprinting analyses. In vitro assays using purified viral proteins will be developed to study methylation and capping of viral mRNAs. In vitro competition assays will be performed to test the hypothesis that viral interference is due to competition between defective interfering particles and standard particles for the viral polymerase. We will try to develop an in vitro replication system so regulation of viral RNA synthesis can be analyzed.